Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)

Abstract

The instant specification provides for evolved base editors which overcome deficiencies of those in art (including increased efficiency and/or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system. In particular, the instant specification provides for evolved cytidine base editors (e.g., based on APOBEC1, CDA, or AID cytidine deaminase domains) which overcome deficiencies of those in art (including increased efficiency and/or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system.

What is claimed is: 
     
       1. A cytidine deaminase comprising an amino acid sequence that is at least 80% identical to amino acid residues 2-208 of SEQ ID NO: 3, wherein the cytidine deaminase comprises one or more mutations selected from the group consisting of H10X 1 , F23X 2 , V75X 3 , K120X 4 , A123X 5 , C158X 6 , I193X 7 , I195X 8 , and V197X 9  relative to SEQ ID NO: 3, or a corresponding mutation(s) in another cytidine deaminase, wherein X 1 , is any amino acid other than H, X 2  is any amino acid other than F, X 3  and X 9  are any amino acid other than V, X 4  is any amino acid other than K, X 5  is any amino acid other than A, X 6  is any amino acid other than C, and X 7  and X8 are any amino acid other than I. 
     
     
       2. The cytidine deaminase of  claim 1  comprising amino acid residues 2-208 of SEQ ID NO: 3, wherein the cytidine deaminase comprises one or more mutations selected from the group consisting of H10X 1 , F23X 2 , V75X 3 , K120X 4 , A123X 5 , C158X 6 , I193X 7 , I195X 8 , and V197X 9  relative to SEQ ID NO: 3, wherein X 1 , is any amino acid other than H, X 2  is any amino acid other than F, X 3  and X 9  are any amino acid other than V, X 4  is any amino acid other than K, X 5  is any amino acid other than A, X 6  is any amino acid other than C, and X 7  and X 8  are any amino acid other than I. 
     
     
       3. The cytidine deaminase of  claim 1 , wherein the cytidine deaminase comprises amino acid residues 2-208 of SEQ ID NO: 7. 
     
     
       4. A fusion protein comprising:
 (i) a nucleic acid programmable DNA binding protein (napDNAbp) and 
 (ii) the cytidine deaminase of  claim 1 . 
 
     
     
       5. A complex comprising the fusion protein of  claim 4  and an RNA bound to the napDNAbp of the fusion protein. 
     
     
       6. A method comprising contacting a nucleic acid molecule with the complex of  claim 5 . 
     
     
       7. A pharmaceutical composition comprising the fusion protein of  claim 4  and a pharmaceutically acceptable excipient. 
     
     
       8. The fusion protein of  claim 4  further comprising a uracil glycosylase inhibitor domain (UGI). 
     
     
       9. The fusion protein of  claim 4 , wherein the amino acid sequence of said fusion protein is the amino acid sequence as set forth in SEQ ID NO: 18.